General Information Regarding Collagen.
Collagen fibrils, proteoglycan aggregates and glycoproteins are critical components of the cartilage extracellular matrix that, collectively, resist compression and the tensile and shear forces that are generated during articulation. Heineg.ang.rd and Oldberg (1989) FASEB J. 3:2042-2051; Mayne and Brewton, Cartilage Degradation: Basic and Clinical Aspects (Woessner, J. F. and Howell, D. S., eds.) Marcel Dekker, Inc., New York, pp. 81-108 (1993). Mutations in cartilage matrix genes or the genes which encode the enzymes that affect the biosynthesis, assembly or interactions between these various matrix components may contribute to degradation of the cartilage matrix and the loss of normal cartilage function.
The Role Of Prolyl-4-Hydroxylase In The Production Of Collagen.
Prolyl-4-hydroxylase plays a crucial role in the synthesis of all collagens. Specifically, the enzyme catalyzes the formation of 4-hydroxyproline in collagens and related proteins by the hydroxylation of proline residues in -Xaa-Pro-Bly-sequences. These 4-hydroxyproline residues are essential for the folding of newly synthesized collagen polypeptide chains into triple-helical molecules.
The vertebrate prolyl-4-hydroxylase is an .alpha..sub.2 .beta..sub.2 tetramer in which the .alpha. subunits contribute to most parts of the catalytic sites. See, Kivirikko, et al., (1989) FASEB J. 3, 1609-1617; Kivirikko, et al., (1990) Ann. N.Y. Acad. Sci. 580, 132-142; Kivirikko, et al., (1992), Post Translational Modifications of Proteins, eds. Harding, J. J. & Crabbe, M. J. C. (CRC, Boca Raton, Fla.), pp. 1-51. The .beta. subunit has been cloned from many sources (id.; see also, Noiva and Lennatz, (1992) J. Biol. Chem. 267:6447-49; Freedman, et al., (1994) Trends Biochem. Sci. 19:331-336) and has been found to be a highly unusual multifunctional polypeptide that is identical to the enzyme protein disulfide-isomerase (Pihlajaniemi, et al. (1987) EMBO J. 6:643-649; Kojvu, et al., (1987) J. Biol. Chem. 262:6447-49), a cellular thyroid hormone-binding protein (Cheng, et al. (1987) J. Biol. Chem. 262:11221-27), the smaller subunit of the microsomal triacylglycerol transfer protein (Wetterau, et al., (1990) J. Biol. Chem. 265:9800-07), and an endoplasmic reticulum luminal polypeptide which uniquely binds to various peptides (Freedman, supra; Noiva, et al. (1991) J. Biol. Chem. 266:19645-649; Noiva, et al. (1993) J. Biol. Chem. 268:19210-217).
A catalytically important .alpha. subunit, designated the .alpha.1 subunit, has been cloned from human (Helaakoski, et al. (1989) Proc. Natl. Acad. Sci. (USA) 86:4392-96), chicken (Bassuk, et al. (1989) Proc. Natl. Acad. Sci. (USA) 86:7382-886) and Caenorhabditis elegans (Veijola, et al. (1994) J. Biol. Chem. 269:26746-753), and its RNA transcripts have been shown to undergo alternative splicing involving sequences encoded by two consecutive, homologous 71-bp exons (Helaakoski, supra; Helaakoski, et al. (1994) J. Biol. Chem. 269:27847-854). A second a subunit, designated the .alpha.2 subunit has been previously obtained from mouse. Helaakoski, et al. (1995) Proc. Natl.Acad.Sci. (USA) 92:4427-4431.